DNA extraction includes the use of an extraction buffer and ethanol:
Procedure
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Procedure: Estimate the number of DNA fragments (our gene of interest, cytochrome b) we may have following extraction.
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We will use the cytochrome b gene (CTYb) for species ID of our bushmeat samples. It meets our requirements. It is shared across all mammals BUT, differs enough between species for the sequence (of As, Ts, Cs and Gs) to give us a species-level ID. |
We would have created PCR cocktails for each of our bushmeat samples and placed into a thermocycler for synthesis. Instead, you will do a short virtual PCR lab. Read over the information in PCR below and complete the procedure.
The PCR mixture is placed inside a thermocycler (PCR machine). It is typically repeated about 35 times and the temperature changes are programmed by researchers and automated by the thermocycler. The process proceeds in three steps as outlined below.
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Procedure
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Extraction of DNA from meat samples is a lengthy and difficult process. It has already been completed for our samples. Today, you will mirror this process: you will be extracting some of your own DNA from your cheek epithelial cells in a Bio @ Home activity. You may need to review the structure of DNA (covered in Ch 4 of your BIOL 120 Pearson text).
Procedure: 3 Selfies
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DNA extraction includes the use of an extraction buffer and ethanol:
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Procedure: Estimate the number of DNA fragments (our gene of interest, cytochrome b) we may have following extraction.
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We will use the cytochrome b gene (CTYb) for species ID of our bushmeat samples. It meets our requirements. It is shared across all mammals BUT, differs enough between species for the sequence (of As, Ts, Cs and Gs) to give us a species-level ID. |
We would have created PCR cocktails for each of our bushmeat samples and placed into a thermocycler for synthesis. Instead, you will do a short virtual PCR lab. Read over the information in PCR below and complete the procedure.
The PCR mixture is placed inside a thermocycler (PCR machine). It is typically repeated about 35 times and the temperature changes are programmed by researchers and automated by the thermocycler. The process proceeds in three steps as outlined below.
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Procedure
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Bushmeat is legal in some African countries but is illegal in Kenya. Once bushmeat as been processed, it is indistinguishable from domestic meat. Therefore DNA analysis is required to determined if the meat sold, labeled as beef, pork, goat or lamb, is actually wildlife meat. We will be testing several samples to ascertain the species of origin.
Please read over the summary below from a report entitled "Lifting the Siege: Securing Kenya's Wildlife."
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Our samples are from the Taita Taveta district of southeastern Kenya, which includes Kenya's largest national park system, Tsavo East and Tsavo West National Parks. Specifically, our samples are from the Kasigau area between the two parks, located on the trailing edge of the Eastern Arc Mountains. The Kasigau landscape is dominated by Mt. Kasigau, which the Titata people settled around to serve as a water catchment. The area also serves as a migration corridor between Tsavo East and West National Parks and is rife with human wildlife conflict.
Over the next several years BIOL 121 students will be testing samples from the five villages that surround Mt. Kasigau: Mwakasinyi, Keteghe, Rukanga, Jora and Bungule. Every semester we will be adding to a bushmeat database, which can be used by conservation groups and the Kenyan Wildife Service to locate hotspots of poaching and bushmeat activity. This semster's samples were sourced from 8 butcheries (view slideshow) from the villages of Rukanga (samples 1-6), Jora (sample 7) and Bungule (sample 8).
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1) Bushmeat Processing |
2) Digestion & Extraction |
3) Polymerase chain reaction (PCR) |
4) Gel electrophoresis |
5) Sequencing |
6) BLASTing |
Lab 10: ProtocolYour task in Lab 10 is to replicate a DNA extraction protocol and determine how much DNA we have successfully extracted form our samples and how much DNA we have following PCR.
Exercise I. How do you extract DNA? Exercise II. Determine How Much DNA We Have After Extraction Exercise III. Determine How Much DNA We Have After PCR |
Exercise II. How much DNA do we have?Now, let's move on to our own samples. Following extraction of DNA from the meat samples, we need to determine how much DNA we have. In other words...we're we successful and to a high enough degree to allow for the next steps?
Proceedure: Estimate the number of DNA fragments (our gene of interest, cytochrome b) we may have by the end of PCR.
1) We start here! Following DNA extraction, we test the amount of purified DNA in our samples, to ensure enough product exists for PCR to work. We conduct this analysis using a spectrophotometer. The results for our samples are shown in the figure at right in ug/ng. Sample 8 yielded no genetic material.
2) How much cytochrome b (cytb) DNA might we have? Using these results calculate the potential total number of cytochrome b (cytb) fragments we have extracted from each sample.
Wait! Why are we looking for the cytochrome b gene? All DNA is NOT the same. Some genes are more conserved than others. The DNA for life, is very similar. For example we share 50% our DNA with bananas. Within our species, we share 99.%. The genes that can be used to distinguish between mammal species are specific. Cytochrome b is one such gene. The sequence of As, Cs, Ts and Gs can tell us from which species it originated. So now our task is to determine out of all the DNA we have extracted, approximately how much if the cytochrome b gene do we have?
You need the following to make your calculations:
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3) Now, let's calculate our PCR yield: how many copies of the cytochrome b gene we may have in each sample following PCR (if all went according to plan). We performed 35 cycles of PCR on our bushmeat samples. Initial denaturation at 94 °C for 4 min and with a final extension at 72 °C for 10 minutes. The equation to calculate the final number of DNA strands created by PCR = N2^n, where N = the original number of DNA molecules to be copied and n = the # of PCR cycles.
4) Add these calculations for each sample to your spreadsheet. |
Part I. Add to our Research LibraryHelp us build a better research library. Please find at least one reference, about bushmeat, Kasigau or Tsavo, or DNA sequencing for species identification.
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Part I. Post Lab AnalysisPlease upload your excel sheet including our DNA concentration calculations. It is NOT complete at this time. That's OK! We'll finish this work in Lab 11!
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